A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining
A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining
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DOI：
10.1002/cyto.a.20582
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发表时间：
2008-07-01
期刊：
影响因子：
3.7
通讯作者：
Moll, Juergen
中科院分区：
文献类型：
--
作者：
Cappella, Paolo;Gasparri, Fabio;Moll, Juergen
定量检测BrdU掺入DNA是一种广泛使用的评估细胞周期状态的技术。BrdU的检测需要DNA变性，缺点是大多数蛋白质表位被破坏，经典的抗体染色技术无法进行多重分析。为了解决这个问题，我们开发了一种新的方法，可以克服DNA变性步骤，但仍然可以检测BrdU。用5-乙炔基-2‘-脱氧尿苷对细胞进行短时间的脉冲作用，这种尿苷被结合到DNA中。然后在生理条件下，DNA中暴露的核苷酸炔基被铜(I)催化的叠氮-炔环加成反应(CuAAC)与BrdU叠氮化合物进行衍生化。随后，商业抗BrdU单抗在不需要变性步骤的情况下检测到与DNA结合的溴代沙星部分。EDU连续标记显示，与BrdU相比，抗增殖活性略有增加。然而，使用较低浓度的EDU进行标记可以弥补这一点。通过使用BrdU叠氮化物的高度特异性反应，可以快速检测到炔基标签。DNA染料PI对两种BrdU叠氮化物的荧光猝灭可以忽略不计。我们的标记方法适用于FCM和HCA，并显示出比其他方法更高的信噪比。该方法还可以通过同时检测EDU-BrdU、caspase-3和磷酸组蛋白3mAb来进行多重分析，证明了这一新技术的敏感性和可行性。此外，它还具有在体内使用的潜力，例如骨髓研究。我们建立了一种新的方法来确定细胞在细胞周期中的位置。这比传统的BrdU检测更优越，因为它允许多重分析，更灵敏，与PI的猝灭更少。该方法为利用脉冲标记实验研究不同细胞周期阶段蛋白质表达的变化提供了新的机会。(C)2008年国际细胞测量促进会。
Quantification of BrdU incorporation into DNA is a widely used technique to assess the cell cycle status of cells. DNA denaturation is required for BrdU detection with the drawback that most protein epitopes are destroyed and classical antibody staining techniques for multiplex analysis are not possible. To address this issue we have developed a novel method that overcomes the DNA denaturation step but still allows detection of BrdU. Cells were pulsed for a short time by 5-ethynyl-2'-deoxyuridine, which is incorporated into DNA. The exposed nucleotide alkyne group of DNA was then derivatized in physiologic conditions by the copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) using BrdU azides. The resulting DNA-bound bromouracil moiety was subsequently detected by commercial anti-BrdU mAb without the need for a denaturation step. Continuous labeling with EdU showed a slightly increased anti-proliferative activity compared to BrdU. However, using a lower concentration of EdU for labeling can compensate for this. Alkynyl tags could be detected quickly by a highly specific reaction using BrdU azides. Fluorescence quenching by the DNA dye PI using both BrdU azides was negligible. Our labeling method is suitable for FCM and HCA and shows a higher signal to noise ratio than other methods. This method also allowed multiplex analysis by simultaneous detection of EdU-BrdU, caspase-3, and phospho-histone 3 mAbs, proving sensitivity and feasibility of this new technique. In addition, it has the potential for use in vivo, as exemplified for bone marrow studies. We have established a new method to determine the position of cells in the cell cycle. This is superior when compared to traditional BrdU detection since it allows multiplex analysis, is more sensitive and shows less quenching with PI. The method provides new opportunities to investigate changes in protein expression at different cell cycle stages using pulse labeling experiments. (c) 2008 International Society for Advancement of Cytometry.